Summary of Work: Three areas of research have been developed recently. In the first set of experiments, the sequence specificity of Rrp1 has been precisely defined with a series of oligonucleotide substrates. These studies demonstrate inhibition of Rrp1 in pyrimidine repeats and at purine-pyrimidine junctions, and a 200-300-fold slower catalytic rate is measured for Rrp1 progressing through repeated thymidine nucleotides compared to repeated adenosine nucleotides. In the future, similar studies of the sequence context effects on the other activities of Drosophila Rrp1 will be carried out. Another area of recent investigation is the level of enzymatic redundancy of DNA repair functions in Saccharomyces cerevisiae. In yeast and other eukaryotes, the identity of all the enzymes involved in the base excision repair pathway are not yet known. Thus, a 3'-phosphodiesterase assay was used to partially purify and characterize a new repair enzyme from yeast, Pde1. Pde1 demonstrates a Mg++-dependent 3'-phosphodieserase with tightly associated class II AP endonuclease, and is likely to regulate genome stability in vivo. The domain structure of Rrp1 is also under study. Previous experiments suggest that Rrp1 has a bipartite structural organization. Proteolysis with V-8 protease separates a carboxy-terminal protease-resistant nuclease domain, confirming the presence of a discrete structural entity. The characteristics of this nuclease domain are currently under study.